Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvii, 154 p.; also includes graphics (some col.) Includes bibliographical references (p. 134-154). Available online via OhioLINK's ETD Center

Thesis (M.S.)--University of Delaware, 2008. / Adviser: Kalmia E. Kniel. Includes bibliographical references.

The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.

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Thesis (Ph. D. - Microbiology and Immunology)--University of Arizona, 1986. / Includes bibliographical references (leaves 121-126).

Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Kalmia Kniel, Dept. of Animal & Food Sciences. Includes bibliographical references.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Medical Virology))--University of Pretoria, 2005. / Medical Virology / unrestricted

Nucleic acid hybridization (gene probe) and polymerase chain reaction (PCR) techniques have been used to detect viral nucleic acid in water. However, gene probe and PCR may not distinguish between infectious and noninfectious viruses. This study evaluated the ability of gene probe to detect viable poliovirus 1 (polio 1), from sterile and nonsterile groundwater, and the ability of PCR to detect infectious human immunodeficiency virus (HIV-1) from tap and wastewater. The plaque forming (BGM cells), and the tissue culture infectious dose fifty (TCID₅₀) (PLC/PRF/5 cells) procedures were used to detect infectious polio 1 and HIV-1, respectively. Detection of polio 1 by gene probe and cell culture was similar in nonsterile water and in filter sterilized water, but not in autoclaved water. These results suggest that in some natural waters, detection of polio 1 by gene probe may correlate to detection by cell culture procedures. Although detection of infectious HIV-1 by cell culture decreased gradually, until no virus could be found, detection by PCR remained positive throughout the study. Therefore, it was concluded that the use of PCR to assess the risk associated to the presence of HIV-1 in polluted waters, may not be adequate. The enteric adenovirus types 40 (Ead 40) and 41 (Ead 41) are considered the second most important cause of viral gastroenteritis in children, but their role as waterborne pathogens is uncertain. This study compared the survival of Ead 40 and Ead 41 with polio 1, and hepatitis A virus (HAV) in different types of water. The Enteric adenoviruses survived longer in tap and sea water than either polio 1 or HAV, but only slightly better in wastewater. These results suggest that the enteric adenoviruses may survive for prolonged periods in water, representing a potential route of transmission. This study evaluated also the concentration of Ead 40 by the filter adsorption-elution method. With negatively-charged filters, recovery efficiencies of 22, 36, and 38% were obtained from secondary sewage, tap and sea water, respectively. Using electropositive filters, Ead 40 was recovered from tap water with an efficiency of 26.5%. These results show that Ead 40 can be concentrated, from water, with an efficiency comparable to that of other enteric viruses.

Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Monitoring water for virus contamination requires two steps: 1) the collection and the concentration of the water sample and 2) the isolation and identification of the virus present. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks, such as: 1) long incubation periods, up to two and three weeks, before some enteric viruses are detected, 2) not all viruses can be detected in one cell line, and 3) not all viruses have been grown in cell culture. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. These techniques also require the production of an antibody for each different virus type. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with ³²P dCTP and ³²P dATP to a specific activity greater then 1.0 x 10⁹ cpm/ug DNA. Gene Screen Plus (NEN) was chosen as the hybridization membrane since it was more sensitive to virus detection than the other membranes tested. A dot-blot apparatus (Bio Rad) was used to apply the samples. Results were visualized by autoradiography for thirty-six hours at -70° C. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tapwater. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses.